Cell culturing device

ABSTRACT

The invention features devices and kits for capturing and culturing microorganisms (e.g., bacteria, fungi, or protists) and methods of using the devices and kits to detect microorganisms in environmental and other samples. The device includes a nutrient media having a flat growth area on which microorganisms can grow. Samples are collected by contacting the device with any environmental sample, e.g., rolling device on a work surface or exposing device to air, or by filtering a sample through a membrane. Microorganisms deposited on the membrane derive nutrients from the underlying media and grow into colonies that can then be detected using methods known in the art. The detected colonies can be imaged digitally or with film.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Application No.61/624,643, filed Apr. 16, 2012, which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

The invention relates to the fields of cell culture and detection.

In many industries, particularly the food, beverage, healthcare,electronic, and pharmaceutical industries, it is essential to analyzesamples rapidly for the degree of contamination by microorganisms, suchas bacteria, yeasts, or molds.

One microbial culture technique, called microbial enumeration or colonycounting, quantifies the number of microbial cells in a sample. Themicrobial enumeration method, which is based on in situ microbialreplication, generally yields one visually detectable “colony” for eachmicrobial cell in the sample. Thus, counting the visible colonies allowsmicrobiologists to accurately determine the number of microbial cells ina sample. To perform microbial enumeration, bacterial cells can bedispersed on the surface of nutrient agar in Petri dishes (“agarplates”) and incubated under conditions that permit in situ bacterialreplication. Microbial enumeration is simple, ultra-sensitive,inexpensive, and quantitative but may also be slow.

There is a need for additional culturing devices and methods for rapidmicrobial enumeration.

SUMMARY OF THE INVENTION

The invention provides devices and kits for capturing and culturingmicroorganisms, e.g., present in environmental samples. In one aspect,the invention provides a cell culturing device including a base thatcontains nutrient media for microorganisms; a porous membrane overlayingthe nutrient media; and a lid that mates with the base to cover themembrane and the nutrient media. The nutrient media has a flat growtharea raised above the base and a circumferential area that slopes fromthe edge of the flat growth area to the base, and the nutrient media iscapable of sustaining growth of microorganisms in the growth area. Incertain embodiments, the base includes polystyrene. The base may alsoinclude a circumferential groove across which the membrane is sealed.The membrane includes, for example, a mixed cellulose ester membrane.The membrane may be substantially non-radiative and substantiallynon-reflective and/or black.

In other embodiments, the growth area has a flatness of 100 to 450 μmbefore collection of a sample or 300 to 500 μm after collection of asample. The nutrient media may or may not sustain growth ofmicroorganisms in the circumferential area. The membrane may be attachedto the base by a film that is sealed to the base. Alternatively, thedevice may further include a film applied to the base andcircumferential area, in which the film adheres the membrane to thebase.

In another aspect the invention provides a cell culturing deviceincluding, a base that contains nutrient media for microorganisms, inwhich the nutrient media has a flat growth area (e.g., a flatness ofabout 100 to 450 microns) raised above the base, in which the nutrientmedia is capable of sustaining growth of microorganisms in the growtharea, a film overlaying the nutrient media, in which the film and thenutrient media have a circumferential area that slopes from the edge ofthe flat growth area to the base, and the film has an opening to exposea portion of the flat growth area, and a lid that mates to the base tocover the nutrient media. In another embodiment, the device alsoincludes a porous membrane in contact with the exposed growth area.

In another aspect, the invention provides a kit for cell culturingincluding a cell culturing device that includes, a base that containsnutrient media for microorganisms, in which the nutrient media has aflat growth area (e.g., flatness of about 100 to 450 microns) raisedabove the base, in which the nutrient media is capable of sustaininggrowth of microorganisms in the growth area, and a film (e.g., anon-porous film) overlaying the nutrient media, in which the film andthe nutrient media have a circumferential area that slopes from the edgeof the flat growth area to the base, and the film has an opening toexpose a portion of the flat growth area, a porous membrane configuredfor placement on the exposed portion of the flat growth area; and a lidthat mates with the base to cover the membrane and the nutrient media.In another embodiment, the film has fiducial marks for placement of amembrane on the growth area. In one embodiment, the kit also includes afiltration device, such as the device in WO 2007/038478, which is herebyincorporated by reference.

In one embodiment of any of the devices or kits of the invention, thelid may include an optically clear material disposed to allow imaging ofthe growth area. The lid when attached to the base may also preventcontamination by ingress of microorganisms, in which the lid isseparated from the membrane by an air gap. The device may include aunique ID label on the base. The ID label may be a bar code or 2Dbarcode. The ID label may be used to track the device identity, devicecompatibility with automated detection instruments and protocols, deviceexpiration date, sterilization history, and other information ofinterest. The device may also include indentations or protrusions toallow for alignment and gripping by human users or instrumentation,e.g., in the lid and/or the device. In certain embodiments, a device isnot compressible in the lateral direction, e.g., to maintain flatness ofthe growth area. In such embodiment, one or more mechanically supportingelements may be incorporated into the device to provide rigidity in thelateral direction. In other embodiments, the device includes a fiducialmark, e.g., of radiative plastic, printed fluorescent material, embossedfluorescent material, or a through hole exposing fluorescent media,material, or plastics, located, e.g., outside the growth area. Fordevices with a separable membrane, the fiducial mark may be a throughhole in the membrane that is located outside of the area through which asample is filtered. Such a through hole may expose fluorescent nutrientmedia, plastic, or printed material. Fiducial marks may be employed toalign a membrane with a growth area and/or in the automated alignment ofmultiple images taken from a device.

In another embodiment, the base mates to the lid to prevent a rotationof greater than about 50 μm of the base relative to the lid.

In another embodiment, the invention provides a device or a kit in whichthe base has a bottom surface and a side wall extending around theperimeter of and upward from the bottom surface, in which the nutrientmedia is within the side wall of the base, and the lid has a top surfaceand a side wall extending around the perimeter of and downward from thetop surface; in which the lid reversibly secures to the base (e.g., thelid secures to the base by axial compression or the lid secures to thebase by rotation of the lid relative to the base).

In another embodiment, the lid or the base also includes acircumferential rim (e.g., continuous or discontinuous) extendinglaterally from the side wall of the base or from the side wall of thelid and a first detent extending laterally from the side wall of thebase or from the side wall of the lid, in which the circumferential rimhas a proximal side facing away from the top of the lid or the bottom ofthe base, a distal side facing toward the top of the lid or the bottomof the base, and a lateral edge connecting the proximal and distalsides, in which the lid has the circumferential rim and the base has atleast one first detent, or the lid has at least one first detent and thebase has the circumferential rim, and in which the lid is secured to thebase by interengagement between the circumferential rim and the at leastone first detent. In another embodiment, at least one first detentextending laterally defines a gap between the first detent and thebottom surface of the base or the top surface of the lid, in which thegap is sized to accept the circumferential rim, and the first detentengages the distal side of the circumferential rim. The first detent mayalternatively engage the lateral side of the circumferential rim. Thedevice may include at least one second detent extending laterally fromthe side wall, in which the circumferential rim includes a kerf, and thesecond detent engages the kerf. Another embodiment of the invention mayinclude a device in which the distal side of the rim is sloped, and theat least one first detent engages the distal side of the rim by relativerotation of the lid to the base. The device may include a plurality ofcircumferential rims and first detents, in which the lid secures to thebase by relative rotation of 90 degrees or less, e.g., 45 degrees orless. The device may include a stop on the lid or base that arrestsrotation of the circumferential rim after a specified amount ofrotation.

In another aspect, the invention provides a kit including a cellculturing device containing a base that contains nutrient media formicroorganisms; and a porous membrane overlaying the nutrient media, inwhich the nutrient media has a flat growth area raised above the baseand a circumferential area that slopes from the edge of the flat growtharea to the base, and in which the nutrient media is capable ofsustaining microorganisms in the growth area; a protective lid attachedto the base and preventing contamination by ingress of microorganisms,in which the protective lid is separated from the membrane by an airgap; and an optical lid attachable to the base when the protective lidis removed and including an optically clear material disposed to allowimaging of the growth area when attached to the base.

In a further aspect, the invention provides a kit including a cellculturing device containing a base that contains nutrient media formicroorganisms, in which the nutrient media has a flat growth arearaised above the base, in which the nutrient media is capable ofsustaining growth of microorganisms in the growth area, a filmoverlaying the nutrient media, in which the film and the nutrient mediahave a circumferential area that slopes from the edge of the flat growtharea to the base, and the film has an opening to expose a portion of theflat growth area, a porous membrane configured for placement on theexposed portion of the flat growth area; a protective lid attached tothe base and preventing contamination by ingress of microorganisms, inwhich the protective lid is separated from the growth area by an airgap; and an optical lid attachable to the base when the protective lidis removed and including an optically clear material disposed to allowimaging of the growth area when attached to the base. The kit may alsoinclude a filtration device as described herein.

The invention also features a method for monitoring the presence ofmicroorganisms by providing a device or kit of the invention; contactingthe growth area of the device with a volume of air or a surface;incubating the device to allow growth of microorganisms; and determiningthe extent of growth of microorganisms. Exemplary surfaces includeindustrial and laboratory surfaces and garments. In certain embodiments,the sample is collected by rolling the device so that thecircumferential area and growth area contact the surface or passing thevolume of air over the growth area. The extent of growth may bedetermined by optically imaging the growth area. An image may beanalyzed to quantify the number of microorganisms. The incubation anddetermining steps may be repeated to determine colonies ofmicroorganisms that grow over time.

In another aspect, the invention provides a method for monitoring thepresence of microorganisms in a sample by providing a device or kit ofthe invention, filtering a sample through the membrane, placing themembrane on the growth area, incubating the device to allow growth ofmicroorganisms; and determining the extent of growth of microorganisms.The extent of growth may be determined by optically imaging the growtharea. An image may be analyzed to quantify the number of microorganisms.The incubation and determining steps may be repeated to determinecolonies of microorganisms that grow over time.

By a “substantially non-radiative” object is meant an object that doesnot emit light, e.g., by fluorescence, phosphorescence, or luminescence.

By a “substantially non-reflective” object is meant an object thatreflects less than 25%, 10%, 5%, 1%, or 0.1% of the light used to imagethe object.

By a “securing member” is meant a component or feature of aspect of amechanical mechanism that joins or affixes two entities. Exemplarysecuring members are threads, catches, detents, rims, latches, hooks,clasps, snaps, bayonet mounts, J-shaped hooks, L-shaped hooks, dents,protrusions, ribs, spring tongues, tabs, grooves, stops, notches, holes,kerfs, compression fits, interference fits, jam fits, cams, and camstops. By “secures” is meant to mate, join or form a union between twoentities in which the rotation of the two entities relative to eachother is limited.

By “circumferential” is meant around the perimeter. Circumferential isnot limited to circular shapes, for the purpose of this invention.

Other features and advantages will be apparent from the followingdescription, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an exploded view of a device of the invention. FIG. 1B is aphotograph of a device with the cover removed.

FIGS. 2A-2B are cross-sectional views of a device of the invention. FIG.2A is a cross-sectional view of a device with a protective lid installedbefore use of the device. FIG. 2B is a cross-sectional view of a devicewith an optical lid installed.

FIGS. 3A-3B are cross-sectional views showing details of attachment of amembrane to a base. FIG. 3A is a cross-sectional view of a part of adevice showing an expansion trough. FIG. 3B is a schematic depiction ofa part of a device showing the location of a film or edge coating andexpansion trough.

FIG. 4 is a depiction of a base of a device of the invention including aregion for application of a bar code, mechanically supporting elements(ribs), a double circumferential side wall, indentations (V features)for alignment and gripping, and a feature for optical alignment.

FIG. 5A is an exploded view of a device of the invention including aprotective lid, a circumferential film with fiducial marks (dashes), abase with nutrient media and a growth area, and a bar code label. FIG.5B is a photograph of a device of the invention including acircumferential film with fiducial marks (dashes), a base with nutrientmedia and a growth area, and a membrane with circumferential fiducialthrough holes overlaying the growth area and partially overlaying thecircumferential film.

FIG. 6 is a schematic depiction of a device of the invention includingan optical lid with a circumferential rim and a base with three firstdetents (Snap detent, Detail A of FIG. 6) and three second detents(Centering detents, Detail B of FIG. 6).

FIG. 7 is a schematic depiction of a device of the invention including alid with a kerfed circumferential rim (Detail D in FIG. 7) and a basewith three first detents (Ball detents, Detail B in FIG. 7), and threethird detents (Centering detents, Detail C in FIG. 7), and nine seconddetents (Detail A in FIG. 7) that are configured to fill within thekerfs.

FIG. 8 is a schematic depiction of a device of the invention including alid having a discontinuous circumferential rim (see Detail A in FIG. 8)and a base with two types of detents that are complementary to thediscontinuous rim of the lid. The detents of the base are separated byabout 40 degrees relative to the circumference. The first detents (seeDetail C of FIG. 8) and second detents (see Detail B of FIG. 8) engagethe discontinuous rim. The lid also has a visual indicator (rimfeature).

FIG. 9 is a schematic depiction of a device of the invention including alid that has two discontinuous, sloping circumferential rims and a basewith three first detents and two stops (Cam Stop, Detail A in FIG. 9).

FIG. 10 is a photograph of a device of the invention including a base, amembrane overlaying a nutrient media, and a circumferential area whichslopes to the base. In addition, fiducial marks (dots) can be seen inthe circumferential area.

FIG. 11 is a photograph of microbial colonies grown on a device andvisible to the human eye.

FIGS. 12A-12C are a set of images showing the use of a device inmonitoring different types of environmental samples: air (FIG. 12A),surfaces such as work areas (FIG. 12B), and garments such as gloves orsleeves (FIG. 12C).

FIG. 13A is an exploded view of a filtration device including a funnel,a membrane with fiducial marks, a mesh support pad, and a filtrationbase. FIG. 13B is a photograph of an assembled filtration device.

FIGS. 14A-14D are a set of images showing the monitoring of a liquidsample using a filtration device and a device of the invention. FIG. 14Ais a photograph of the assembled filtration device, a protective lid,and a device of the invention. FIG. 14B is a photograph of filtering asample through the membrane within the filtration device. FIG. 14C is aphotograph of the disassembled filtration device, showing the funnel,membrane on the filtration base, protective lid, and a device of theinvention. FIG. 14D is a photograph showing the transfer of the membraneto a device of the invention, in which the membrane is placed with thefiducial marks on the circumferential rim and fiducial marks(circumferential through holes) in the membrane allow for alignment ofmultiple images take of the membrane.

DETAILED DESCRIPTION OF THE INVENTION

The invention features devices and kits for capturing and culturingmicroorganisms (e.g., bacteria, fungi, or protists) and methods of usingthe devices and kits to detect microorganisms in environmental samples.The device is useful for rapid environmental monitoring and can be usedto collect microorganisms, for example, by rolling the device on asurface. The device is then incubated to allow any microorganismscollected to grow into colonies, which are indicative of microbialcontamination.

Device

The cell culturing devices of the invention facilitate the samplecollection, sample growth, and detection of microorganisms within asample. Devices of this invention allow for efficient, cost effective,and robust microorganism monitoring for a wide variety of applications.

The device, e.g., as shown in (FIGS. 1A-1B), includes a base thatcontains nutrient media overlaid by a porous membrane. The membrane isin conformal contact with the nutrient media. The membrane and nutrientmedia form a flat growth area surrounded by a sloping circumferentialarea. In preferred embodiments, the membrane allows nutrients to passthrough and sustain microbial growth in the growth area but not in thecircumferential area. In one aspect of the invention, the device isuseful for automated detection of microorganisms, e.g., using the GrowthDirect™ system for rapid colony counting, e.g., as described in U.S.Publication No. 2003/0082516, which is hereby incorporated by reference.The device may be of any appropriate size, e.g., that of a RODAC™ plate.

The base may be substantially non-radiative and non-reflective and maybe made of any suitable material, e.g., polystyrene or other plastic.The base may also have a circumferential groove (also referred to as anexpansion trough) that can be used for attachment of a membrane (e.g.,as shown in FIGS. 2 and 3). The base may be produced by methods known inthe art, e.g., injection molding.

The device, e.g., the base and/or the lid, may include indentations(e.g., as shown in FIG. 4) or protrusions for gripping or alignment onthe device. In certain embodiments, the device is not compressible inthe lateral direction. For example, the device may include one or moremechanically supporting elements (FIG. 4) to maintain the flatness ofthe growth area during handling.

The device includes a porous membrane, e.g., one having fluorescenceproperties commensurate with detection of autofluorescent microbialmicrocolonies. For example, the membrane is substantially non-radiativeand non-reflective for detection of autofluorescent microbialmicrocolonies. Membranes may be manufactured from materials includingcellulose, cellulose acetate, polystyrene, polyethylene, polycarbonate,polyethylene terephthalate (PET), polyolefin, ethylene vinyl acetate,polypropylene, polysulfone, polytetrafluoroethylene, nylon, and siliconecopolymer. The choice of membrane depends, in part, on the type of cellto be cultured (e.g., microorganisms that grow attached to a surface(anchorage-dependent), microorganisms that grow in suspension(anchorage-independent), or microorganisms that grow as attached to asurface or in suspension), degree of permeability, and rate of transferof fluids and gases. An exemplary membrane is a black mixed celluloseester membrane (Sartorius AG).

The membrane is placed over the nutrient media so that the membrane andmedia are in conformal contact. The membrane and nutrient media form aflat growth area raised above the base with a circumferential slopingarea around the edges. Such a design makes the device suitable forcontact testing, e.g., by rolling the device on a surface. The membraneand nutrient media form a growth area that is flat across an area, e.g.,of 10, 15, 20, 25, 30, 35, or 50 cm², preferably at least at least 25cm². The membrane on the nutrient media has a flatness of about 100 to600 μm, e.g., 200 to 350 μm, e.g., about 300 μm, as fabricated or 300 to500 μm, e.g., about 450 μm, after collection of sample. The membrane ispreferably factory installed and stays wet for the life of the product.The membrane has pores so that microorganisms deposited on the membranemay obtain nutrients from the underlying nutrient media. Examples ofmembrane pore sizes are 0.45 μm and 0.22 μm.

Solid or semi-solid nutrient growth media can be employed in the presentdevice. Examples include Sabouraud dextrose agar (SDA), R2A agar,tryptic soy agar (TSA) letheen, and plate count agar (PCA). The mediamay be poured onto the base in a molten liquid state and then allowed tosolidify into a flat growth area that is raised above the base and acircumferential area that slopes from the edge of the flat growth areato the base. The flatness of the growth area may be controlled bysurface tension and by filling normal to gravity. The flatness of thegrowth area may also be achieved using several alternate methods. Forexample, one method to achieve a flat growth area includes pouringmolten nutrient media onto the underside of a pre-attached, wetmembrane. In this alternate method, the membrane is pre-attached to abase that has an opening on the bottom. The opening is used to fillmolten nutrient media, e.g., agar. This opening is then sealed postfilling by a cover or film. The membrane is circumferentially sealed toprevent leakage. The membrane expands or inflates during the fillingprocess and may be shaped by trapping within a nest or cavity ofappropriate shape. Another method to achieve flatness is to pre-bow thebase mid section downward by approximately 150 to 200 μm, e.g., usingvacuum. The nutrient media is poured, and, once the media solidifieswith a concave surface, the bowing force is released and the growth areasprings back to the flat state. Alternatively, the nutrient media may bea liquid media held in a porous matrix, which is shaped to have a flatgrowth area and sloping circumferential area.

The membrane is preferably secured to prevent peeling during use. Themembrane may be installed by heat sealing to the base, e.g., by bridgingover a circumferential groove. The groove (FIG. 3A) allows expansion ofthe membrane without wrinkling. The groove also traps any moistureformed during the attachment. Alternatively, the membrane may beattached to the device using a circumferential film, e.g., of PET,co-PET, acetate, or polyimide, that may be used to define the edges ofthe growth area. The film may be made black. The film may besubstantially non-radiative and/or non-reflective. The film may containseveral layers (FIG. 3B). Such layers allow for heat fusion to themembrane without damaging the membrane, low fluorescence, and/or lowreflectance. A film may also cover the circumferential area to preventgrowth of microorganisms outside the flat growth area. This arrangementis beneficial in confining growth to an area of the device, e.g., forimaging or visual inspection.

In alternative design, a device does not include an integral membrane.This device also includes a nutrient media raised above the base, a flatgrowth area, a film overlaying the circumferential area of the nutrientmedia (FIGS. 5A and 5B), and the circumferential area that slopes fromthe edge of the flat growth area to the base. The device includes a filmthat overlays the circumferential area (in whole or in part), and thefilm has an opening to expose a portion of the flat growth area. Theopening in the film and the nutrient media form a growth area that isflat across an area, e.g., of 10, 15, 20, 25, 30, 35, or 50 cm²,preferably at least at least 25 cm². Suitable films include anynon-porous or hydrophobic plastic or material, e.g., PET, co-PET,acetate, or polyimide. The film may be made black. The film may besubstantially non-radiative and/or non-reflective. The growth areaprovides a surface where a porous membrane, e.g., exposed, to a samplemay be overlaid. The nutrient media provides sustained growth oforganisms on the top surface of the overlaid membrane, thereby providinga device for cell culturing and detection of contamination.

The device also includes a lid. The lid is for example a protective lid(FIG. 2A) that does not contact the membrane and prevents contaminationof the device prior to use. The lid may also be an optical lid (FIG. 2B)with an optically clear window for imaging the growth area, e.g., withvisible or UV light. An exemplary material for the lid is Zeonor® 1060R(polycycloolefin resin; Zeon Chemicals LP). Glass may also be employed.The optical lid may also be used as a protective lid prior to use. Thelid mates to the base to seal the membrane and nutrient media fromoutside contamination. The lid may be produced by methods known in theart, e.g., injection molding. All or part of the lid may benon-radiative and/or non-reflective.

A lid secures to the base using securing members present on both the lidand the base. The lid and base may secure or engage reversibly, in whichthe lid and base may be separated and reattached multiple times.Securing the lid to the base affixes the lid relative to the base in theaxial direction (z-axis), thereby sealing the device. Securing membersmay provide alignment of the lid relative to the base securing in thelateral directions (x-axis and y-axis). Preferably, the lid protects thebase and also prevents rotation of the lid relative to the base, e.g.,to less than 50 μm.

A lid may be secured to the base with axial compression. For example, acircumferential rim (FIG. 6) on the lid and detents (snap detents, FIG.6, Detail A) on the base may be used to secure the lid to the base oncean axial force is applied to the lid. The detent is deflected as thecircumferential rim passes the detent. The circumferential rim rests ina gap between the snap detent and the bottom surface of the base. Thesnap detent (Detail A of FIG. 6) may have a pointed protrusion to reducethe contacting surface area of the snap detent, thus increasing theforce per area applied to the circumferential rim. In addition to thesnap detents, centering detents can be used that apply pressure to thelateral edge of a circumferential rim (Detail B of FIG. 6), thus fixingthe position of the lid. The reproducibility of the optical lid positionrelative to the base is important for improving the consistency ofautomated sample imaging. Securing and preventing movement of theoptical lid improves the reproducibility of the automated imageanalysis.

In an alternative embodiment, the base may include multiple ball detents(Detail B of FIG. 7), multiple second detents (Detail A of FIG. 7), andmultiple centering detents, (Detail C of FIG. 7), around perimeter ofthe base. The lid is secured to the base first detent by acircumferential rim (FIG. 7). The second detents help align the lidrelative to the base. The kerfed circumferential rim (Detail D of FIG.7) prevents rotation of the lid relative to the base because the seconddetents (Detail A of FIG. 7) fill the kerfs of the rim.

A lid may be secured to the base with a rotational motion of less thanor equal to 90 degrees. For example, the lid may be have a series ofdiscontinuous circumferential rims (FIG. 8, Detail A). The discontinuouscircumferential rims may or may not be evenly distributed around theperimeter, each about 40 degrees apart (FIG. 8). A series of detents maybe located on the base (FIG. 8). Two variations of detents are shown(Detail C and Detail B of FIG. 8), but a single type may be employed.The first detent engages to the top edge (distal edge adjacent to topsurface of the lid) of the circumferential rim in the portion oppositethe vertical portion of the discontinuous circumferential rim. Thesecond detent engages to the top edge (distal edge adjacent to topsurface of the lid) of the circumferential rim.

A lid may also be secured to the base with a rotational motion of lessthan or equal to 90 degrees. For example, the base includes multipledetents (Detail A of FIG. 9) sized and shaped to reversibly secure to adiscontinuous, sloped circumferential rim (FIG. 9). The first detent hasa horizontal length with a top surface that prevents lid from beinginstalled in wrong location. The first detent facilitates a jam fit byproviding an upper boundary that increasingly interacts with thevariable height (sloped) discontinuous circumferential as the lid istwisted relative to the base. A stop (cam stop, Detail A of FIG. 9)provides a rotational boundary for the discontinuous circumferentialrim. The increasing height of the circumferential rim engages the firstdetent as the rim is turned about 90 degrees, thereby inducing a jamfit. The cam stop prevents over-rotation, providing a stop for theleading edge of the circumferential rim.

Additional non-limiting exemplary securing members and mechanisms forsecuring a lid to the base include: threads, clamps, gaskets, magnets,crown caps, and friction fits. For example, a lid of the device of theinvention may be configured with a series of threads. A base of theinvention may be configured with a complimentary series of threads, forsecuring the lid to the base.

The protective lid and the optical lid may attach to the same base usingthe same or different mechanisms. The securing members may be on the lidor the base. For example, the circumferential rim may be on the lid, andthe detents on the base. Alternatively, the detents may be on the lid,and the circumferential rim on the base. Circumferential rim may also beon the outer or inner perimeter of the side wall of the lid or the base.

The device may include features that indicate successful securing of thelid to the base. For example, a rim feature on a lid (FIG. 8) may beabove a base and be visible before the lid is secured to the base. Oncethe lid is secured to the base, the rim feature is not visible above thebase, thereby indicating a successful union of the lid and the base. Thedevice may include securing members compatible with automation. Forexample, a securing member may be sized and shaped such that robotictransfer arms may engage a securing member, thereby facilitating theautomated use of the device.

The device may also include a fiducial mark, e.g., printed fluorescentmaterial, embossed fluorescent material, radiative plastic, or a throughhole exposing fluorescent media, material, or plastics. Other fiducialmarks are known in the art. The fiducial mark may be outside the growtharea (FIG. 5B and FIG. 10). The fiducial mark may help align a membranewith the device (FIG. 5B). A fiducial mark may be used to align multipleimages acquired from the same device, e.g., taken at different times, orthe fiducial mark may be used to align the device within an imager. Thefiducial marks may be at any position on the device that is suitable forimaging, e.g., the base, the circumferential area, the growth area, orthe lid.

The device may also have a unique ID label imprinted or affixed on thedevice to aid in automated handling or sample tracking, e.g., by theGrowth Direct™ system. The ID label may be a bar code or 2D barcode. TheID label may be used to track the device identity, device compatibilitywith instruments and protocols, device expiration date, sterilizationhistory, and other information of interest.

Methods of Use

The devices can be used to monitor the presence of microorganisms, e.g.,in the environment. Environmental samples may include, withoutlimitation, air, surfaces, and garments. The devices and kits of theinvention may be used in any situation where microbial contaminationneeds to be rapidly detected, e.g., laboratories, hospitals,manufacturing areas, and “clean rooms” for nanotechnology manufacturingand applications. Exemplary surface samples include surfaces ofstainless steel, glass or granite work surfaces, walls, floors andequipment surfaces. Surface may also include anatomical structures suchas fingers and foreheads. Exemplary garment samples include jacketsleeves, gloves, chest plate and any other portion of wearable garment.The method may include: contacting the growth area of the device with avolume of air or a surface; incubating the device to allow growth ofmicroorganisms (incubation may occur at, above, or below roomtemperature); and determining the extent of growth of microorganisms,e.g., by manual counting or by automated counting of colonies (as shownin FIG. 11). Any culturable microorganism, including bacteria,cyanobacteria, protozoa, and fungi may be employed in conjunction withthe device described herein. The device can be used for aerobic andanaerobic testing.

The sample may be collected by rolling the device so that thecircumferential area and the growth area contact the surface. Thesurface can be, e.g., work surfaces such as a laboratory surface orindustrial surface (FIG. 12B), garments (FIG. 12C), or gloves (FIG.12C). In another aspect of the invention, air samples can be tested bypassing a volume of air over the growth area, e.g., in an air monitor(FIG. 12A) or by leaving the device uncovered for a period of time.

After sample collection, the device is typically covered using theoptical cover and is incubated for microorganisms to grow, e.g., in anincubator at temperatures above or below room temperature. In oneembodiment, after sample collection, the device is placed within theGrowth Direct™ system for incubation and imaging. The device may beimaged at predefined intervals of time, and microorganisms may bedetected by suitable methods known in the art, e.g., fluorescence (viaautofluorescence or stains), reflectance, or absorbance. Alternatively,vital stains may be introduced into the nutrient media and absorbed intothe microorganisms during growth. Detection may be repeated to discerngrowing colonies from non-growing microorganisms or debris. Images ofmicroorganisms may be recorded, either digitally or with film. Theoptical lid of the device may be removed manually or using automationand replaced with a protective lid during storage. The protective lidmay reversibly secure the base using one or more securing members. Anoptical lid may reversibly secure the base using one or more securingmembers.

Alternatively the sample may be collected by filtering a sample througha membrane and then applying the membrane to the device of theinvention. For example, a membrane and filtration device (FIGS. 13A and13B, and FIG. 14A) may be used to filter a liquid sample, thuscollecting any contaminating microorganisms on the membrane. As shown inFIGS. 13A-13B, an exemplary filtration device includes a funnel thattraps a membrane to a filtration base. The membrane is supported by amesh support pad to maintain flatness during filtration. Otherfiltration devices are known in the art, e.g., as described in WO2007/038478. A sample of interest is passed through the membrane tocollect microorganisms (FIG. 14B). The membrane is removed from thefiltration device and overlaid onto the nutrient media of a deviceconfigured to accept a membrane (FIGS. 14C-14D). The membrane may bealigned relative to the fiducial marks on the film (dashes). Themembrane may include fiducial marks, e.g., through holes in the regionoutside of the filtered area. Such through holes can be used to exposefluorescent media, plastic, or printed material that is otherwisecovered by the membrane. The membrane may be larger diameter than theexposed growth area and overlap the circumferential film. The membranemay adhere to the growth area by wetting from the nutrient. The membraneand device are then covered, incubated, and imaged as described above.

Other methods and instruments for manual or automated colony countingthat can be used with the device are known in the art.

Kit for Environmental Monitoring and Filtering

The invention also features a kit which includes the device, aprotective lid, and an optical lid. The kit may be shipped with aprotective cover installed on the device. The device with the protectivecover and the optical cover may be packaged separately or together insterile packaging. In use, the protective cover is removed, the samplingis done, and the optical cover is then installed.

Alternatively, the invention also features a kit which includes thedevice, a protective lid, a membrane, and an optical lid. The kit may beshipped with a protective cover installed on the device. The device withthe protective cover, membrane and the optical cover may be packagedseparately or together in sterile packaging. In use, the protectivecover is removed, the sampling is done, and the optical cover is theninstalled. Such kits may also include a filtration device as discussedherein.

Other Embodiments

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims.

What is claimed is:
 1. A cell culturing device comprising: (a) a basethat contains nutrient media for microorganisms; (b) a porous membraneoverlaying the nutrient media, wherein the nutrient media has a flatgrowth area raised above the base and a circumferential area that slopesfrom the edge of the flat growth area to the base, and wherein thenutrient media is capable of sustaining growth of microorganisms in thegrowth area; and (c) a lid that mates with the base to cover themembrane and the nutrient media.
 2. The device of claim 1, wherein thegrowth area has a flatness of about 100 to 450 microns before collectionof a sample or about 300 to 600 microns or less after collection of asample.
 3. The device of claim 1, wherein the membrane is not porous inthe circumferential area or the membrane is attached to the base by afilm that is sealed to the base.
 4. The device of claim 1, furthercomprising a film applied to the base and circumferential area, whereinthe film adheres the membrane to the base.
 5. A cell culturing devicecomprising: (a) a base that contains nutrient media for microorganisms,wherein the nutrient media has a flat growth area raised above the base,wherein the nutrient media is capable of sustaining growth ofmicroorganisms in the growth area, (b) a film overlaying the nutrientmedia, wherein the film and the nutrient media have a circumferentialarea that slopes from the edge of the flat growth area to the base, andthe film has an opening to expose a portion of the flat growth area, and(c) a lid that mates with the base to cover the nutrient media.
 6. Thedevice of claim 5, further comprising a porous membrane releasably incontact with the exposed growth area.
 7. A kit for cell culturingcomprising: (a) a cell culturing device comprising: (i) a base thatcontains nutrient media for microorganisms, wherein the nutrient mediahas a flat growth area raised above the base, wherein the nutrient mediais capable of sustaining growth of microorganisms in the growth area,and (ii) a film overlaying the nutrient media, wherein the film and thenutrient media have a circumferential area that slopes from the edge ofthe flat growth area to the base, and the film has an opening to exposea portion of the flat growth area; (b) a porous membrane configured forplacement on the exposed portion of the flat growth area; and (c) a lidthat mates with the base to cover the membrane and the nutrient media.8. The device of claim 5, wherein the growth area has a flatness ofabout 100 to 450 microns.
 9. The device of claim 5, wherein the film isnot porous or the film has fiducial marks for placement of a membrane onthe growth area.
 10. The device of claim 6, wherein the membrane hasthrough holes that expose fluorescent nutrient media, plastic, orprinted material.
 11. The device of claim 6, wherein the membranecomprises a mixed cellulose ester membrane.
 12. The device of claim 6,wherein the membrane is substantially non-radiative and substantiallynon-reflective or black.
 13. The device of claim 5, wherein the basecomprises polystyrene.
 14. The device of claim 5, further comprising afiducial mark.
 15. The device of claim 14, wherein the fiducial mark isoutside the growth area.
 16. The device of claim 15, wherein thefiducial mark comprises radiative plastic, printed fluorescent material,or embossed fluorescent material.
 17. The device of claim 5, wherein thelid comprises an optically clear material disposed to allow imaging ofthe growth area.
 18. The device of claim 5, wherein the lid whenattached to the base prevents contamination by ingress ofmicroorganisms, and wherein the lid is separated from the membrane by anair gap.
 19. The device of claim 5, wherein the base mates to the lid toprevent a rotation of greater than about 50 μm of the base relative tothe lid.
 20. The device of claim 5, wherein the base has a bottomsurface and a side wall extending around the perimeter of and upwardfrom the bottom surface, wherein the nutrient media is within the sidewall of the base, and the lid has a top surface and a side wallextending around the perimeter of and downward from the top surface;wherein the lid reversibly secures to the base.
 21. The device of claim20, wherein the lid secures to the base by axial compression or byrotation of the lid relative to the base.
 22. The device of claim 20,further comprising a circumferential rim extending laterally from theside wall of the base or from the side wall of the lid and a firstdetent extending laterally from the side wall of the base or from theside wall of the lid, wherein the circumferential rim has a proximalside facing away from the top of the lid or the bottom of the base, adistal side facing toward the top of the lid or the bottom of the base,and a lateral edge connecting the proximal and distal sides, wherein thelid has the circumferential rim and the base has at least one firstdetent, or the lid has at least one first detent and the base has thecircumferential rim, and wherein the lid is secured to the base byinterengagement between the circumferential rim and the at least onefirst detent.
 23. The device of claim 22, wherein the circumferentialrim is continuous around the side wall of the base or the side wall ofthe lid.
 24. The device of claim 22, wherein the at least one firstdetent extending laterally defines a gap between the first detent andthe bottom surface of the base or the top surface of the lid, whereinthe gap is sized to accept the circumferential rim, and the first detentengages the distal side of the circumferential rim.
 25. The device ofclaim 22, wherein the at least one first detent engages the lateral sideof the circumferential rim.
 26. The device of claim 22, furthercomprising at least one second detent extending laterally from the sidewall, wherein the circumferential rim comprises a kerf, and the seconddetent engages the kerf.
 27. The device of claim 22, wherein thecircumferential rim is not continuous along the circumference of theside wall of the base or the lid.
 28. The device of claim 27, whereinthe distal side of the rim is sloped, and the at least one first detentengages the distal side of the rim by relative rotation of the lid tothe base.
 29. The device of claim 28, further comprising a plurality ofcircumferential rims and first detents, wherein the lid secures to thebase by relative rotation of 90 degrees or less.
 30. The device of claim28, further comprising a stop on the lid or base that arrests rotationof the circumferential rim after a specified amount of rotation.
 31. Amethod for monitoring the presence of microorganisms in the environment,the method comprising the steps of: (a) providing a device of claim 1;(b) contacting the growth area of the device with a volume of air or asurface; (c) incubating the device to allow growth of microorganisms;and (d) determining the extent of growth of microorganisms.
 32. A methodfor monitoring the presence of microorganisms in a sample, the methodcomprising the steps of: (a) providing a device of claim 5 and a porousmembrane; (b) filtering a sample through the membrane; (c) placing themembrane on the growth area; (d) incubating the device to allow growthof microorganisms; and (e) determining the extent of growth ofmicroorganisms.
 33. The method of claim 32, wherein step (e) comprisesoptically imaging the growth area.
 34. The method of claim 33, furthercomprising analyzing the image to quantify the number of microorganisms.35. The method of claim 33, further comprising repeating steps (d) and(e) to determine growing colonies of microorganisms.
 36. A kitcomprising: (a) a cell culturing device comprising: i) a base thatcontains nutrient media for microorganisms, and ii) a porous membraneoverlaying the nutrient media, wherein the nutrient media has a flatgrowth area raised above the base and a circumferential area that slopesfrom the edge of the flat growth area to the base, and wherein thenutrient media is capable of sustaining microorganisms in the growtharea; (b) a protective lid attached to the base and preventingcontamination by ingress of microorganisms, wherein the protective lidis separated from the membrane by an air gap; and (c) an optical lidattachable to the base when the protective lid is removed and comprisingan optically clear material disposed to allow imaging of the growth areawhen attached to the base.
 37. A kit for cell culturing comprising: (a)a cell culturing device comprising: i) a base that contains nutrientmedia for microorganisms, wherein the nutrient media has a flat growtharea raised above the base, wherein the nutrient media is capable ofsustaining growth of microorganisms in the growth area, and ii) a filmoverlaying the nutrient media, wherein the film and the nutrient mediahave a circumferential area that slopes from the edge of the flat growtharea to the base, and the film has an opening to expose a portion of theflat growth area, (b) a porous membrane configured for placement on theexposed portion of the flat growth area; (c) a protective lid attachedto the base and preventing contamination by ingress of microorganisms,wherein the protective lid is separated from the growth area by an airgap; and (d) an optical lid attachable to the base when the protectivelid is removed and comprising an optically clear material disposed toallow imaging of the growth area when attached to the base.
 38. Thedevice of claim 1, wherein the nutrient media is not capable ofsustaining growth of microorganisms in the circumferential area.
 39. Thekit of claim 36, wherein the nutrient media is not capable of sustaininggrowth of microorganisms in the circumferential area.